Chlorophyll is a green-coloured pigment widely found throughout the plant kingdom. Chlorophyll is essential for photosynthesis and is one of the most abundant organic metal compounds found on earth. Thus many products derived from plants, including foods and feeds, contain significant amounts of chlorophyll.
For example, vegetable oils derived from oilseeds such as soybean, palm or rape seed (canola), cotton seed and peanut oil typically contain some chlorophyll. However the presence of high levels of chlorophyll pigments in vegetable oils is generally undesirable. This is because chlorophyll imparts an undesirable green colour and can induce oxidation of oil during storage, leading to a deterioration of the oil.
Various methods have been employed in order to remove chlorophyll from vegetable oils. Chlorophyll may be removed during many stages of the oil production process, including the seed crushing, oil extraction, degumming, caustic treatment and bleaching steps. However the bleaching step is usually the most significant for reducing chlorophyll residues to an acceptable level. During bleaching the oil is heated and passed through an adsorbent to remove chlorophyll and other colour-bearing compounds that impact the appearance and/or stability of the finished oil. The adsorbent used in the bleaching step is typically clay.
In the edible oil processing industry, the use of such steps typically reduces chlorophyll levels in processed oil to between 0.02 to 0.05 ppm. However the bleaching step increases processing cost and reduces oil yield due to entrainment in the bleaching clay. The use of clay may remove many desirable compounds such as carotenoids and tocopherol from the oil. Also the use of clay is expensive, this is particularly due to the treatment of the used clay (i.e. the waste) which can be difficult, dangerous (prone to self-ignition) and thus costly to handle. Thus attempts have been made to remove chlorophyll from oil by other means, for instance using the enzyme chlorophyllase.
In plants, chlorophyllase (chlase) is thought to be involved in chlorophyll degradation and catalyzes the hydrolysis of an ester bond in chlorophyll to yield chlorophyllide and phytol. WO 2006009676 describes an industrial process in which chlorophyll contamination can be reduced in a composition such as a plant oil by treatment with chlorophyllase. The water-soluble chlorophyllide which is produced in this process is also green in colour but can be removed by an aqueous extraction or silica treatment.
Chlorophyll is often partly degraded in the seeds used for oil production as well as during extraction of the oil from the seeds. One common modification is the loss of the magnesium ion from the porphyrin (chlorin) ring to form the derivative known as pheophytin (see FIG. 1). The loss of the highly polar magnesium ion from the porphyrin ring results in significantly different physico-chemical properties of pheophytin compared to chlorophyll. Typically pheophytin is more abundant in the oil during processing than chlorophyll. Pheophytin has a greenish colour and may be removed from the oil by an analogous process to that used for chlorophyll, for instance as described in WO 2006009676 by an esterase reaction catalyzed by an enzyme having a pheophytinase activity. Under certain conditions, some chlorophyllases are capable of hydrolyzing pheophytin as well as chlorophyll, and so are suitable for removing both of these contaminants. The products of pheophytin hydrolysis are the red/brown-colored pheophorbide and phytol. Pheophorbide can also be produced by the loss of a magnesium ion from chlorophyllide, i.e. following hydrolysis of chlorophyll (see FIG. 1). WO 2006009676 teaches removal of pheophorbide by an analogous method to chlorophyllide, e.g. by aqueous extraction or silica adsorption.
Pheophytin may be further degraded to pyropheophytin, both by the activity of plant enzymes during harvest and storage of oil seeds or by processing conditions (e.g. heat) during oil refining (see “Behaviour of Chlorophyll Derivatives in Canola Oil Processing”, JAOCS, Vol, no. 9 (September 1993) pages 837-841). One possible mechanism is the enzymatic hydrolysis of the methyl ester bond of the isocyclic ring of pheophytin followed by the non-enzymatic conversion of the unstable intermediate to pyropheophytin. A 28-29 kDa enzyme from Chenopodium album named pheophorbidase is reportedly capable of catalyzing an analogous reaction on pheophorbide, to produce the phytol-free derivative of pyropheophytin known as pyropheophorbide (see FIG. 1). Pyropheophorbide is less polar than pheophorbide resulting in the pyropheophoribe having a decreased water solubility and an increased oil solubility compared with pheophorbide.
Depending on the processing conditions, pyropheophytin can be more abundant than both pheophytin and chlorophyll in vegetable oils during processing (see Table 9 in volume 2.2. of Bailey's Industrial Oil and Fat Products (2005), 6th edition, Ed. by Fereidoon Shahidi, John Wiley & Sons). This is partly because of the loss of magnesium from chlorophyll during harvest and storage of the plant material. If an extended heat treatment at 90° C. or above is used, the amount of pyropheophytin in the oil is likely to increase and could be higher than the amount of pheophytin. Chlorophyll levels are also reduced by heating of oil seeds before pressing and extraction as well as the oil degumming and alkali treatment during the refining process. It has also been observed that phospholipids in the oil can complex with magnesium and thus reduce the amount of chlorophyll. Thus chlorophyll is a relatively minor contaminant compared to pyropheophytin (and pheophytin) in many plant oils.
Each of the four chlorophyll derivatives, chlorophyll a and b and pheophytin a and b, exist as a pair of epimers determined by the stereochemistry of H and COOCH3 around the carbon number 132 (numbering according to the IUPAC system, marked with asterisk in FIG. 2). Thus chlorophyll a exists as the pair of epimers chlorophyll a and chlorophyll a′, and chlorophyll b comprises b and b′ forms. Likewise pheophytin a comprises the epimer a and a′ pair and pheophytin b comprises b and b′ forms. The prime (′) forms have S-stereochemistry and non-prime forms have R-stereochemistry about the carbon 132 atom. Epimerization of, for example, the a form to a′ form and vice versa can take place under certain conditions via a common enol, as described in “Epimerization in the pheophytin a/a′ system”, Chemistry letters (1984), 1411-1414. In solution there is typically an equilibrium which dictates the distribution of prime and non-prime chlorophyll compounds and this is often determined by physical parameters such as temperature, pH, solvent and so on.
In general enzymes typically act as stereospecific catalysts by having activity on only one stereoisomer. Previous analyses suggested that chlorophyllases possess a high degree of stereospecificity only catalyzing the hydrolysis of non-prime forms of chlorophyll compounds (see “The stereospecific interaction between chlorophylls and chlorophyllase” J. Biol. Chem. 267(31):22043-22047 (1992)).
In methods for the removal of chlorophyll and chlorophyll derivatives from plant oil which employ chlorophyllases or related enzymes, the stereospecificity of the enzyme may be problematic. In particular, depending on the distribution and equilibrium of the chlorophyll stereoisomers in the oil, a complete degradation of chlorophyll components can be very difficult. For instance, if a significant proportion of the chlorophyll or chlorophyll derivative exists in the prime form, this fraction of the chlorophyll derivatives present in the oil may be resistant to enzymatic degradation. Moreover, a number of enzymes show much lower activity on pyropheophytin than on, for example, pheophytin.
This problem with existing methods is illustrated in FIG. 3. FIG. 3 shows the epimerization of pheophytin a and the conversion to pyropheophytin. The pH of a water/crude plant oil mixture (e.g. comprising about 1-2% water) is typically around 5.0 at about 60° C. Under such conditions, in crude soy bean or rape seed oil the pheophytin a epimer distribution is typically around 70% pheophytin a (R-stereoisomer) and 30% pheophytin a′ (S-stereoisomer) and isomerization between the two epimers is slow. Moreover, variable amounts of pyropheophytin may be formed depending on reaction conditions. If the enzyme used in the reaction is predominantly active only on pheophytin a, a significant proportion of chlorophyll derivatives present in the oil cannot be hydrolyzed directly by the enzyme at unmodified pH.
There is a therefore a need for an improved process for removing chlorophyll and chlorophyll derivatives such as pheophytin and pyropheophytin from plant oils. In particular, there is a need for a process which enhances the removal of various forms of chlorophyll and chlorophyll derivatives from the oil.